This section is intended to introduce the reader to various aspects of art that may be related to various aspects of the present invention, which are described and/or claimed below. This discussion is believed to be helpful in providing the reader with background information to facilitate a better understanding of various aspects of the present invention. Accordingly, it should be understood that these statements are to be read in this light, and not as admissions of prior art.
Tau proteins are proteins that stabilize microtubules. They are abundant in neurons in the central nervous system. When tau proteins are defective, and do not stabilize microtubules properly, they can result in tauopathies. Tauopathies are neurodegenerative diseases resulting from the pathological aggregation of tau protein in the human brain. The best known tauopathy is Alzheimer's disease (“AD”). Some other tauopathies include certain forms of frontotemporal lobar degeneration (e.g., Pick's disease, progressive supranuclear palsy, corticobasal degeneration, etc.) and chronic traumatic encephalopathy.
AD is the most common form of dementia, and is a terminal neurodegenerative disease that lasts about 8 years from diagnosis to death [Hebert L E, Scherr P A, Bienias J L, Bennett D A, Evans D A (2003) Alzheimer disease in the US population: prevalence estimates using the 2000 census. Arch Neurol 60:1119-1122; Thies W, Bleiler L (2011) 2011 Alzheimer's disease facts and figures. Alzheimers Dement 7:208-244]. It was first described by German psychiatrist and neuropathologist Alois Alzheimer in 1906 and was named after him. AD is a progressive neurodegenerative disorder primarily affecting brain regions associated with memory, judgment, and other higher cognitive functions. It is the leading cause of dementia in the world, with a current prevalence of over 5 million cases in the U.S. alone. Most often, it is diagnosed in people over 65 years of age, although the less-prevalent early-onset AD can occur much earlier. Owing to the aging population, this number is expected to triple in the coming decades. Thus, AD will continue to be a major source of morbidity and a driver of health care costs in the U.S. as it affects nearly one in three seniors>80 years of age.
Although the course of AD is unique for every individual, there are many common symptoms. The earliest observable symptoms are often mistakenly thought to be ‘age-related’ concerns, or manifestations of stress. In the early stages, the most common symptom is inability to acquire new memories, observed as difficulty in recalling recent events. When AD is suspected, the diagnosis is usually confirmed with behavioral assessments and cognitive tests, often followed by a brain scan.
As the disease advances, symptoms include confusion, irritability and aggression, mood swings, language breakdown, long-term memory loss, and the general withdrawal of the sufferer as their senses decline. Gradually, bodily functions are lost, ultimately leading to death. Individual prognosis is difficult to assess, as the duration of the disease varies. AD develops for an indeterminate period of time before becoming fully apparent, and it can progress undiagnosed for years.
Evidence gathered to date suggests that AD is primarily a sporadic disease, with genetic mutations accounting for only a small percentage of cases. As a result, identifying the causes of disease onset and progression in an aging but otherwise normal population remains among the highest priorities in biological science. Traditionally, the search for clues begins with disease pathology since AD is definitively diagnosed only upon autopsy. AD is characterized by the presence of two types of neuropathological hallmarks: neurofibrillary tangles (NFTs) and senile plaques. NFTs are intraneuronal aggregates of the microtubule associated protein tau. They are formed by hyperphosphorylation of tau protein, causing it to aggregate in an insoluble form. These aggregations of hyperphosphorylated tau protein may also be referred to as “paired helical filaments” (PHF). The precise mechanism of tangle formation is not completely understood, and whether tangles are a primary causative factor in the disease or play a more peripheral role is still a matter of debate. Senile plaques are extracellular and are primarily composed of amyloid β-peptide. The mechanism through which these lesions develop and the methods needed to detect them in living cases has remained obscure since their discovery more than 100 years ago.
The mechanisms that drive tau lesion formation in the highly prevalent sporadic form of AD are not fully understood, but appear to involve abnormal post-translational modifications (PTMs) that influence tau function, stability, and aggregation propensity. For example, hyperphosphorylation of tau protein on certain hydroxy-amino acids favors lesion formation by dissociating tau from its microtubule binding partner [Biernat J, Gustke N, Drewes G, Mandelkow E M, Mandelkow E (1993) Phosphorylation of Ser262 strongly reduces binding of tau to microtubules: distinction between PHF-like immunoreactivity and microtubule binding. Neuron 11:153-163; Bramblett G T, Goedert M, Jakes R, Merrick S E, Trojanowski J Q, Lee V M (1993) Abnormal tau phosphorylation at Ser396 in Alzheimer's disease recapitulates development and contributes to reduced microtubule binding. Neuron 10:1089-1099] and by directly raising its rate and extent of aggregation [Alonso A, Zaidi T, Novak M, Grundke-Iqbal I, Iqbal K (2001) Hyperphosphorylation induces self-assembly of tau into tangles of paired helical filaments/straight filaments. Proc Natl Acad Sci USA 98:6923-6928, Esteve P O, Chang Y, Samaranayake M et al (2011) A methylation and phosphorylation switch between an adjacent lysine and serine determines human DNMT1 stability. Nat Struct Mol Biol 18:42-48; Necula M, Kuret J (2004) Pseudophosphorylation and glycation of tau protein enhance but do not trigger fibrillization in vitro. J Biol Chem 279:49694-49703]. Although tau phosphorylation state is mediated directly by phosphotransferases, it also is modulated by competing modifications on hydroxylamino acids such as O-linked β-N-acetylglucosaminylation (O-GlcNAcylation) [Liu F, Iqbal K, Grundke-Iqbal I, Hart G W, Gong C X (2004) O-GlcNAcylation regulates phosphorylation of tau: a mechanism involved in Alzheimer's disease. Proc Natl Acad Sci USA 101:10804-10809]. The reciprocal relationship between these tau modifications is leveraged by O-GlcNAcase inhibitors, which by increasing O-GlcNAcylation, lower phosphorylation stoichiometry and depress neurofibrillary lesion formation [Yuzwa S A, Vocadlo D J (2009) O-GlcNAc modification and the tauopathies: insights from chemical biology. Curr Alzheimer Res 6:451-454]. In addition to hydroxy amino acids, Lys residues are modified on tau protein, and these too can influence tau metabolism and aggregation. For example, ubiquitylation of tau at Lys residues modulates intracellular tau levels [Petrucelli L, Dickson D, Kehoe K et al (2004) CHIP and Hsp70 regulate tau ubiquitination, degradation and aggregation. Hum Mol Genet. 13:703-714, Shimura H, Schwartz D, Gygi S P, Kosik K S (2004) CHIP-Hsc70 complex ubiquitinates phosphorylated tau and enhances cell survival. J Biol Chem 279:4869-4876], the magnitude of which affects both nucleation and extension phases of the aggregation reaction [Congdon E E, Kim S, Bonchak J, Songrug T, Matzavinos A, Kuret J (2008) Nucleation-dependent tau filament formation: the importance of dimerization and an estimation of elementary rate constants. J Biol Chem 283:13806-13816]. Together these observations suggest that tau aggregation is under complex regulatory control that involves crosstalk among diverse and sometimes competing PTMs.
Presently, there are no easy, straightforward, noninvasive, definitive methods to diagnose Alzheimer's disease. Biopsy samples from a subject could be used to definitively diagnose AD, but risks include possible anesthetic complications, hemorrhage, infections or seizures [Schuette A J, Taub J S, Hadjipanayis C G, Olson J J (2010) Open biopsy in patients with acute progressive neurologic decline and absence of mass lesion. Neurology 75:419-424, Warren J D, Schott J M, Fox N C et al (2005) Brain biopsy in dementia. Brain 128:2016-2025]. For this reason, AD is currently determined in living subjects by review of medical history, administration of a panel of neuropsychiatric examinations, and structural CT and/or MRI imaging (to eliminate the possibility of vascular dementia, normal pressure hydrocephalus, subdural hematoma or solid tumor) [Knopman D S, DeKosky S T, Cummings J L et al (2001) Practice parameter: diagnosis of dementia (an evidence-based review). Report of the Quality Standards Subcommittee of the American Academy of Neurology. Neurology 56:1143-1153; McKhann G, Drachman D, Folstein M, Katzman R, Price D, Stadlan E M (1984) Clinical diagnosis of Alzheimer's disease: report of the NINCDS-ADRDA Work Group under the auspices of Department of Health and Human Services Task Force on Alzheimer's Disease. Neurology 34:939-944]. This method is at best 80% sensitive and 70% specific [Knopman D S, DeKosky S T, Cummings J L et al (2001) Practice parameter: diagnosis of dementia (an evidence-based review). Report of the Quality Standards Subcommittee of the American Academy of Neurology. Neurology 56:1143-1153]. AD currently is only definitively diagnosed at autopsy. However, NFTs appear long before death, and even decades before the onset of dementia [Braak H, Braak E (1991) Neuropathological stageing of Alzheimer-related changes. Acta Neuropathol 82:239-259], offering the opportunity to detect disease and intervene in disease progression at a much earlier stage than is possible today. Furthermore, because NFT load correlates with neurodegeneration [Gomez-Isla T, Price J L, McKeel D W, Jr., Morris J C, Growdon J H, Hyman B T (1996) Profound loss of layer II entorhinal cortex neurons occurs in very mild Alzheimer's disease. J Neurosci 16:4491-4500] and cognitive decline in AD [Ghoshal N, Garcia-Sierra F, Wuu J et al (2002) Tau conformational changes correspond to impairments of episodic memory in mild cognitive impairment and Alzheimer's disease. Exp Neurol 177:475-493], direct pre mortem detection could help monitor the effectiveness of drug treatments over time [Small G W, Bookheimer S Y, Thompson P M et al (2008) Current and future uses of neuroimaging for cognitively impaired patients. Lancet Neurol 7:161-172], while improving the performance and cutting the costs of clinical trials.
To capture changes in tau burden in living cases, powerful immunoassays, namely enzyme-linked immunosorbent assay (ELISA), capable of detecting minute quantities of tau analytes in cerebral spinal fluid (CSF) have been developed. However, the molecular characterization of tau in CSF presents an analytical challenge for several reasons. First, in the adult human brain there are six different tau isoforms produced from a single gene by alternative mRNA splicing. This heterogeneity is compounded by extensive post-translational modifications. Second, there is relatively low concentration of tau in CSF, ranging from approximately 300 ng/L in healthy individuals to approximately 900 ng/L in AD-afflicted individuals. Considering that this quantity is distributed over many differentially modified forms and six splice variants, the amount available for analysis of each molecular species falls close to the detection limit of most assays. Nevertheless, the first study in which total tau was successfully analyzed in CSF was published in 1995, demonstrating that total tau (t-tau) concentration was significantly elevated in AD patients compared to other neurodegenerative disorders [Arai H, Terajima M, Miura M et al (1995) Tau in cerebrospinal fluid: a potential diagnostic marker in Alzheimer's disease. Ann Neurol 38:649-652; Blennow K, Wallin A, Agren H, Spenger C, Siegfried J, Vanmechelen E (1995) Tau protein in cerebrospinal fluid: a biochemical marker for axonal degeneration in Alzheimer disease? Mol Chem Neuropathol 26:231-245].
Initial studies used antibodies insensitive to the modification status of the protein, thereby measuring the t-tau protein concentration. Of the more than 50 studies conducted on AD patients and controls to date, almost all have shown an increase in t-tau in AD patients by approximately 300% with a sensitivity and specificity of 80-90% [Blennow K, Hampel H (2003) CSF markers for incipient Alzheimer's disease. Lancet Neurol 2:605-613; Blennow K, Zetterberg H (2009) Cerebrospinal fluid biomarkers for Alzheimer's disease. J Alzheimers Dis 18:413-417; Hampel H, Burger K, Teipel S J, Bokde A L, Zetterberg H, Blennow K (2008) Core candidate neurochemical and imaging biomarkers of Alzheimer's disease. Alzheimers Dement 4:38-48; Shaw L M, Vanderstichele H, Knapik-Czajka M et al (2009) Cerebrospinal fluid biomarker signature in Alzheimer's disease neuroimaging initiative subjects. Ann Neurol 65:403-413]. However, t-tau has significantly greater discriminative power in the young (<70 years old) compared to the old (>70 years old) [Burger nee Buch K, Padberg F, Nolde T et al (1999) Cerebrospinal fluid tau protein shows a better discrimination in young old (<70 years) than in old old patients with Alzheimer's disease compared with controls. Neurosci Lett 277:21-24]. As some phosphorylated motifs (p-tau) are characteristic of AD, by using antibodies specific to such modifications, these assays have been greatly improved. For example p-tau231 and p-tau181 can be used to distinguish AD from control groups and even from other neurological conditions, including frontotemporal lobar degeneration, dementia with Lewy bodies, vascular dementia, and major depression [Bian H, Van Swieten J C, Leight S et al (2008) CSF biomarkers in frontotemporal lobar degeneration with known pathology. Neurology 70:1827-1835, Buerger K, Zinkowski R, Teipel S J et al (2003) Differentiation of geriatric major depression from Alzheimer's disease with CSF tau protein phosphorylated at threonine 231. Am J Psychiatry 160:376-379; Grossman M, Farmer J, Leight S et al (2005) Cerebrospinal fluid profile in frontotemporal dementia and Alzheimer's disease. Ann Neurol 57:721-729; Hampel H, Buerger K, Zinkowski R et al (2004) Measurement of phosphorylated tau epitopes in the differential diagnosis of Alzheimer disease: a comparative cerebrospinal fluid study. Arch Gen Psychiatry 61:95-102; Hampel H, Teipel S J (2004) Total and phosphorylated tau proteins: evaluation as core biomarker candidates in frontotemporal dementia. Dement Geriatr Cogn Disord 17:350-354; Vanmechelen E, Vanderstichele H, Davidsson P et al (2000) Quantification of tau phosphorylated at threonine 181 in human cerebrospinal fluid: a sandwich ELISA with a synthetic phosphopeptide for standardization. Neurosci Lett 285:49-52]. CSF p-tau levels correlate with cognitive decline in patients with mild cognitive impairment (MCI) [Buerger K, Teipel S J, Zinkowski R et al (2002) CSF tau protein phosphorylated at threonine 231 correlates with cognitive decline in MCI subjects. Neurology 59:627-629] and with neocortical NFT-pathology in AD [Buerger K, Ewers M, Pirttila T et al (2006) CSF phosphorylated tau protein correlates with neocortical neurofibrillary pathology in Alzheimer's disease. Brain 129:3035-3041]. Furthermore both t-tau and p-tau predict rate of cognitive decline in different stages of AD [Blom E S, Giedraitis V, Zetterberg H et al (2009) Rapid progression from mild cognitive impairment to Alzheimer's disease in subjects with elevated levels of tau in cerebrospinal fluid and the APOE epsilon4/epsilon4 genotype. Dement Geriatr Cogn Disord 27:458-464; Buerger K, Ewers M, Andreasen N et al (2005) Phosphorylated tau predicts rate of cognitive decline in MCI subjects: a comparative CSF study. Neurology 65:1502-1503; Samgard K, Zetterberg H, Blennow K, Hansson O, Minthon L, Londos E (2010) Cerebrospinal fluid total tau as a marker of Alzheimer's disease intensity. Int J Geriatr Psychiatry 25:403-410] and concentration of p-tau231 declined longitudinally from mild to moderate AD [Hampel H, Buerger K, Kohnken R et al (2001) Tracking of Alzheimer's disease progression with cerebrospinal fluid tau protein phosphorylated at threonine 231. Ann Neurol 49:545-546] and correlated significantly at baseline with rate of hippocampal atrophy in mild to moderate AD, acting as an indicator of structural disease progression [Hampel H, Burger K, Pruessner J C et al (2005) Correlation of cerebrospinal fluid levels of tau protein phosphorylated at threonine 231 with rates of hippocampal atrophy in Alzheimer disease. Arch Neurol 62:770-773]. In a recent European multi-center-study, CSF p-tau reliably predicted AD in subjects with MCI with high accuracy (80%) as a single biomarker in a relatively short but clinically relevant observation interval of 1.5 years [Ewers M, Buerger K, Teipel S J et al (2007) Multicenter assessment of CSF-phosphorylated tau for the prediction of conversion of MCI. Neurology 69:2205-2212]. Combination of this biomarker with t-tau and Aβ42 can be used with optimized accuracy to detect incipient AD in subjects with MCI with positive and negative predictive values of >80% [Hansson O, Zetterberg H, Buchhave P, Londos E, Blennow K, Minthon L (2006) Association between CSF biomarkers and incipient Alzheimer's disease in patients with mild cognitive impairment: a follow-up study. Lancet Neurol 5:228-234; Herukka S K, Hallikainen M, Soininen H, Pirttila T (2005) CSF Abeta42 and tau or phosphorylated tau and prediction of progressive mild cognitive impairment. Neurology 64:1294-1297; Mattsson N, Zetterberg H, Hansson O et al (2009) CSF biomarkers and incipient Alzheimer disease in patients with mild cognitive impairment. JAMA 302:385-393; Zetterberg H, Wahlund L O, Blennow K (2003) Cerebrospinal fluid markers for prediction of Alzheimer's disease. Neurosci Lett 352:67-69], thus addition of unique epitopes specific to pathological tau may provide further accuracy and sensitivity to already established methods of diagnosis.
However, the understanding of the PTMs is not complete, and so accuracy of the current diagnostic tests could still be improved. Further, even though some diagnostic methods have been developed (with limited success), there is still no method for locating and removing abnormally modified tau from the body.